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首页> 外文期刊>Journal of Molecular Biology >Guiding ribozyme cleavage through motif recognition: The mechanism ofcleavage site selection by a group II intron ribozyme
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Guiding ribozyme cleavage through motif recognition: The mechanism ofcleavage site selection by a group II intron ribozyme

机译：通过基序识别指导核酶切割：II组内含子核酶切割位点的机制

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摘要

The mechanism by which group II introns cleave the correct phosphodiester linkage was investigated by studying the reaction of mutant substrates with a ribozyme derived from intron ai5 gamma. While fidelity was found to be quite high in most cases, a single mutation on the substrate (+1C) resulted in a dramatic loss of fidelity. When this mutation was combined with a second mutation that induces a bulge in the exon binding site 1/intron binding site 1 (EBS1/IBS1) duplex, the base-pairing register of the EBS1/IBS1 duplex was shifted and the cleavage site moved to a downstream position on the substrate. Conversely, when mismatches were incorporated at the EBS1/IBS1 terminus, the duplex was effectively truncated and cleavage occurred at an upstream site. Taken together, these data demonstrate that the cleavage site of a group II intron ribozyme can be tuned at will by manipulating the thermodynamic stability and structure of the EBS1/IBS1 pairing. The results are consistent with a model in which the cleavage site is not designated through recognition of specific nucleotides (such as the 5'-terminal residue of EBS1). Instead, the ribozyme detects a structure at the junction between single and double-stranded residues on the bound substrate. This finding explains the puzzling lack of phylogenetic conservation in ribozyme and substrate sequences near group II intron target sites.
机译：通过研究突变型底物与内含子ai5γ衍生的核酶的反应，研究了II组内含子切割正确的磷酸二酯键的机理。尽管在大多数情况下保真度很高，但底物上的单个突变（+ 1C）导致保真度急剧下降。当此突变与诱导外显子结合位点1 /内含子结合位点1（EBS1 / IBS1）双链体凸起的第二个突变结合时，EBS1 / IBS1双链体的碱基配对寄存器被移位，切割位点移至基板上的下游位置。相反，当在EBS1 / IBS1末端掺入错配时，双链体被有效地截短，并且在上游位点发生切割。综上所述，这些数据表明，可以通过操纵EBS1 / IBS1配对的热力学稳定性和结构来随意调节II型内含子核酶的切割位点。该结果与其中未通过识别特定核苷酸（例如EBS1的5'-末端残基）指定切割位点的模型相符。相反，核酶在结合的底物上的单链和双链残基之间的连接处检测结构。这一发现解释了II组内含子靶位点附近的核酶和底物序列中缺乏令人困惑的系统发育保守性。

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